Kidney vetch (Anthyllis vulneraria L.) is an old medicinal plant, which is mostly known from ethnomedicine. Nowadays, its herbal parts are only scarcely used in phytotherapy, whereas medicinal applications of root parts have not been reported so far. In this study we investigated ethyl acetate extracts, obtained from A. vulneraria roots. A completely different metabolite profile compared to that of aerial parts was detected by HPLC-ESI-(HR)MSn , mainly prenylated chalcones, flavones, isoflavones and cinnamic acids . Two main chalcones (23 and 24) were isolated from the EtOAc extracts by preparative chromatography. Based on 1D- and 2D-NMR data 24 was assigned to the known compound kanzonol C, while 23 as a novel chalcone. Moreover, anti-proliferative activities of the EtOAc extract and of isolated chalcones were investigated in different liver, breast, and thyroid carcinoma cell lines. Interestingly, the EtOAc extract and 24 exhibited the most pronounced decrease of cell viability in the MDA-MB468 breast carcinoma cell line with an IC50 value of 5.97 µM. Two synthetic derivatives of 24 (hexahydro- and triacetyl kanzonol, 30, 31, respectively), 23 and isobavachalcone 18 were less active . Thus, the prenylated chalcones in A. vulneraria roots appear to be promising lead structures for further anti-proliferative studies.